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Arch Pathol Lab Med—Vol 133, April 2009 Breast Carcinoma After Neoadjuvant Therapy —Sahoo & Lester 633

Review Article

Pathology of Breast Carcinomas AfterNeoadjuvant Chemotherapy

An Overview With Recommendations on Specimen Processing and Reporting

Sunati Sahoo, MD; Susan C. Lester, MD, PhD

● Context.—Currently, more women are being treated withchemotherapy or hormonal agents before surgery (neoadjuvant chemoendocrine therapy) for earlier-stage operablebreast carcinoma. The pathologic examination of thesespecimens can be quite challenging.

Objective.—To give an overview of (1) pathologic chang-

es that occur during treatment, (2) systems for evaluatingresponse to treatment, and (3) recommendations for path-ologic examination and reporting of such cases.

Data Sources.—The recommendations are based on thereview of selected literature on breast carcinoma after

neoadjuvant therapy and the authors’ personal experiencewith the clinical and pathologic characteristics of casesfrom each of the authors’ own institutions.

Conclusions.—Pathologists play a key role in the evalu-ation of pathologic response, which is extremely importantas a prognostic factor for individual patients, as a short-

term endpoint for clinical trials, and as an adjunct for re-search studies. Therefore, surgical pathologists must be fa-miliar with the gross examination, sampling, and reportingof breast carcinomas after neoadjuvant therapy.

(Arch Pathol Lab Med. 2009;133:633–642)

BACKGROUND AND SCOPE

Neoadjuvant therapy (NAT), also termed primary orpreoperative therapy, refers to the treatment of patientswith systemic agents before definitive surgical removal of a carcinoma. It was originally used as a standard treat-ment for inflammatory and inoperable locally advanced breast cancers.1,2 Diagnosis was typically made by fine-

needle aspiration. Therefore, pathologic information aboutthe carcinoma (eg, type and grade) and information forstaging (eg, size and lymph node status) was limited. Thecombination of image-guided core needle biopsy, sentinellymph node biopsy (or fine-needle aspiration of a palpableor suspicious node), and new breast imaging modalities(such as magnetic resonance imaging) have substantiallyincreased the ability to accurately classify and stage car-cinomas before surgical excision. Thus, increasing num- bers of women with earlier-stage operable breast cancersare now being treated with chemotherapy or hormonalagents before surgery.3–5 Recently, a detailed review on‘‘Preoperative Therapy in Invasive Breast Cancer: Review-ing the State of the Science and Exploring New ResearchDirections’’ was conducted at the 2007 National CancerInstitute State of the Science meeting. The materials pre-

Accepted for publication August 7, 2008.From the Department of Pathology, Brigham and Women’s Hospital,

Boston, Mass (Dr Lester); and the Department of Pathology, Universityof Louisville, Louisville, Ky (Dr Sahoo).

The authors have no relevant financial interest in the products orcompanies described in this article.

Reprints: Sunati Sahoo, MD, Department of Pathology, University of Louisville, 530 S Jackson Street, Louisville, KY 40202 (e-mail: [email protected]).

sented at this meeting are publicly available at http://ctep.cancer.gov/bcmeeting.

Published clinical trials have shown that systemic ther-apy before or after surgery gives identical results for lo-coregional control and metastasis-free survival.2,3,6 How-ever, the following are several major advantages of neoadjuvant therapy.

1. The efficacy of systemic therapy can be assessed invivo. The response, or nonresponse, of cancers can be de-termined in individual patients. In all studies, comparedto poor responders, patients who achieve a good clinicalresponse have improved long-term disease-free survivaland overall survival, indicating that clinical response can be used as an early surrogate of outcome. Patients whoachieve a pathologic complete response (pCR), that is, noresidual invasive carcinoma in the breast or lymph nodes,have an excellent prognosis.2,7–15 Information about re-sponse can also be used to modify or change treatmentfor patients whose carcinomas show little or no changeafter initial treatment.

2. Tumor response is a short-term endpoint for clinicaltrials. After a breast carcinoma is surgically removed, thenext endpoint for a clinical trial is recurrence or death.However, these events may not occur for many years oreven decades. In addition, if a patient does well, it cannot be determined if this outcome is due to the effectivenessof the systemic treatment or because the carcinoma wascompletely removed by surgery. Tumor response providesimportant clinical information within months, which canallow more rapid assessment of new treatments with few-er patients.

3. There is increased eligibility for breast conservation.In most studies of NAT, most carcinomas are reduced in

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634 Arch Pathol Lab Med—Vol 133, April 2009 Breast Carcinoma After Neoadjuvant Therapy —Sahoo & Lester

size, and some women are able to conserve their breastinstead of requiring mastectomy.16

4. Research linking tumor response to tissue samples isfacilitated. In many trials, tissue samples are collected be-fore, during, and after treatment. Tumor samples pairedwith information about tumor response are powerful re-search tools for identifying techniques to predict response,as well as for understanding how and why tumors re-spond to therapy. Such studies have identified gene ex-pression profiles associated with tumor response.17–20

Here we describe the (1) pathologic changes in breastcancers after treatment, (2) systems for evaluating re-sponse to treatment, and (3) recommendations for patho-logic examination and reporting.

PATHOLOGIC CHANGES OCCURRING INCARCINOMAS DUE TO NEOADJUVANT THERAPY

Although there are many different combinations of agents used for NAT, typical changes are seen in mostcarcinomas with any type of treatment. The followingchanges are commonly observed after neoadjuvant ther-apy.

Tumor Size

Neoadjuvant therapy reduces the size of the primarytumor for most patients. Size can usually be determined by palpation in patients with a minimal treatment re-sponse. However, tumors that have undergone a markedresponse are more difficult to palpate. The reason for thechange in palpability is secondary to marked softening of the tumor stroma, the quality of the desmoplastic re-sponse, and changes in cellularity. In addition, a decreasein blood flow, as frequently detected by magnetic reso-nance imaging, could contribute to a change in the firm-ness of the carcinoma. In cases of a marked or completeresponse, it may not be possible to identify the originaltumor site (tumor bed) by gross examination.

CellularityCarcinomas typically become less cellular after treat-

ment, even when there is not a marked decrease in size.Loss of cellularity correlates with clinical response andprognosis.10,21 For tumors that remain as a contiguousmass, cellularity can be estimated over the entire carci-noma. Estimates of cellularity are more difficult whenthere has been a marked response, because islands of highly cellular carcinoma may be interspersed within alarge, difficult-to-delineate tumor bed. Since carcinomas before treatment also vary greatly in cellularity, a changein cellularity is easier to determine if the pretreatment car-cinoma is available for comparison.

Histologic Appearance and Tumor Grade

Most carcinomas do not change in appearance aftertreatment, except for the loss of cellularity. However, sometumors may appear to be higher grade, and in rare in-stances may be of lower grade because of the cytomor-phologic changes seen in the residual tumor cells fromtreatment effect.2,22,23 A change in tumor grade can only beassessed by comparing the posttreatment tumor to thepretreatment biopsy before attributing the cellular pleo-morphism to treatment effect. The cytologic effects of treatment resulting in a change in the grade of cancershave not been clearly correlated with clinical outcome, and

it is not yet known if this will be an important prognosticfactor. For unknown reasons, in some tumors, the in situcarcinoma and tumor emboli in vascular spaces (lympho-vascular invasion or LVI) are relatively resistant to treat-ment when compared to carcinoma invading the stroma.22

For a detailed description of the treatment-inducedchanges, see the ‘‘Microscopic Appearance of Tumor Bed’’in the section titled, ‘‘Pathologic Examination AfterNeoadjuvant Therapy.’’

Tumor MarkersIn general, tumor markers remain the same before and

after treatment.24 In rare instances of discrepancies, labo-ratory error in testing, interpretation of the stains, tumorsampling (eg, there may be little tumor to evaluate either before or after treatment) and tumor heterogeneity must be considered. However, in some cases, a change in tumormarker expression may result directly from specific typesof neoadjuvant therapy. For example, progesterone recep-tor is frequently lost after treatment with aromatase in-hibitors, but not with tamoxifen.25 HER2/neu expressionrarely changes after chemotherapy,26 but may be dimin-ished in a subset of carcinomas after treatment with tras-tuzumab (Herceptin; Genentech, South San Francisco, Cal-

if).27

It is unknown if this change is due to downregulationor selection of tumor cells not expressing HER2/neu. In arecent study by Tacca et al,28 98 (23%) of 420 patients whoreceived NAT had a change in hormone receptor status onrepeat immunohistochemical studies. In this study, 61 of 145 patients (42%) who were initially receptor negative became receptor positive. The hormone receptor–positiveswitch was significantly correlated with better overall sur-vival in these patients, compared with patients with un-changed hormone receptor–negative tumors.

Changes in Ki-67 (MIB-1) have been suggested as ameans to measure response to therapy, particularly withhormonal therapy where inhibition of proliferation is theprimary goal, though hormonal agents have the capabilityof promoting cell death. Complete pathologic responses

are uncommon after such treatment and changes in pro-liferation have not yet been linked to survival after hor-monal treatment.25

Response in Lymph Nodes

Lymph node status is the most important prognosticfactor in patients who receive neoadjuvant therapy. Eval-uation of treatment response in lymph nodes is more com-plicated and may not be possible in certain patients, as theonly involved nodes might have been removed at the timeof sentinel node biopsy before NAT. Thus, it is preferableto evaluate clinically palpable or sonographically abnor-mal nodes with fine-needle aspiration rather than with biopsy if NAT is planned. Patients who have negative sen-

tinel node biopsy before receiving NAT usually do notundergo axillary dissection (ie, they are assumed to benode negative).

The response in the breast and in the lymph node isgenerally similar. Patients with complete response in both breast and axillary lymph nodes have significantly im-proved overall and disease-free survival.9 In fact, the prog-nostic significance of a pathologic complete response inthe breast is marginal or absent in models that control forpathologic responses in lymph nodes.8,9,14,29

Not all of a patient’s lymph node metastases will re-spond equally to chemotherapy. Some nodal metastases

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Table 1. Criteria Used in Different Systems forCategorizing Response to Treatment*

NSABP B-182

Category

pCR No recognizable invasive tumor cells presentpPR The presence of scattered individual or small

clusters of tumor cells in a desmoplastic or hy-aline stroma

pNR Tumors not exhibiting the changes listed above

Miller-Payne System10

Grade 1 No change or some alteration to individual ma-lignant cells, but no reduction in overall cellu-larity (pNR)

Grade 2 A minor loss of tumor cells, but overall cellulari-ty still high; up to 30% loss (pPR)

Grade 3 Between an estimated 30% and 90% reductionin tumor cells (pPR)

Grade 4 A marked disappearance of tumor cells such thatonly small clusters or widely dispersed indi-vidual cells remain; 90% loss of tumor cells(almost pCR)

Grade 5 No malignant cells identifiable in sections fromthe site of the tumor; only vascular fibroelas-totic stroma remains, often containing macro-phages; however, ductal carcinoma in situmay be present (pCR)

Chevallier Method7

Class 1 Disappearance of all tumor (pCR)Class 2 Presence of DCIS in the breast, no invasive car-

cinoma and negative lymph node (pCR)Class 3 Presence of invasive carcinoma with stromal al-

teration (pPR)Class 4 Few modifications of the tumoral appearance

(pNR)

Sataloff Method8

Tumor

T-A Total or near total therapeutic effect (pCR)T-B 50% therapeutic effect, but less than total or

near total (pPR)T-C 50% therapeutic effect, but effect evident (pPR)T-D No therapeutic effect (pNR)

Nodes

N-A Evidence of therapeutic effect, no metastatic dis-ease

N-B No nodal metastasis or therapeutic effectN-C Evidence of therapeutic effect, but nodal metas-

tasis presentN-D Viable metastatic disease, no therapeutic effect

RCB System15

RCB-0 No carcinoma in breast or lymph node (pCR)RCB-I Partial responseRCB-II Partial responseRCB-III Chemoresistant

AJCC ‘‘y’’ Classification12

Category

T Uses same criteria as before treatmentN Uses same criteria as before treatment

* NSABP indicates National Surgical Adjuvant Breast and Bowel Pro- ject; pCR, pathologic complete response; pPR, pathologic partial re-sponse; pNR, pathologic no response; DCIS, ductal carcinoma in situ;RCB, residual cancer burden; and AJCC, American Joint Committee onCancer.

with good response may not leave any evidence of priortumor involvement or may show fibrous scarring with lit-tle or no residual carcinoma, whereas other nodes mayhave large metastases after treatment.

The significance of the size of a metastatic deposit in alymph node depends on whether or not the patient has been treated. In the National Surgical Adjuvant Breast andBowel Project (NSABP) B-18, at 9 years of follow-up, pa-tients with negative nodes or micrometastases, who werenot treated with chemotherapy before surgery, had iden-

tical survival, whereas those patients with macrometas-tases had a significantly worse survival.2 However, afterNAT, survival of patients with minimetastases (1.0 mm)and micrometastases (2.0 mm) in lymph nodes was sim-ilar to that of patients with macrometastases and signifi-cantly worse than that of patients with negative nodes.2

Thus, the prognostic significance of small lymph node me-tastases after neoadjuvant therapy, particularly in a lymphnode with prior greater involvement, is different whenthese same findings are present in the node of a patientwho has not received NAT.2,30 Micrometastases in lymphnodes in patients who receive NAT probably representmacrometastases that have partially responded to che-motherapy.

SYSTEMS FOR EVALUATING PATHOLOGIC RESPONSETO TREATMENT

The same prognostic factors evaluated for untreated car-cinomas are important for carcinomas after treatment, butmay be modified by changes caused by the treatment. Sev-eral studies have attempted to provide criteria for patho-logic response after treatment (Table 1). Although the cri-teria for classifying pathologic response have not beenstandardized, in most studies the degree of response totreatment has been shown to correlate with survival (Table2). In general, all of the systems recognize a category of pathologic complete response (pCR) and a category of lit-tle or no response. The number of categories of partialresponse in different systems ranges from 1 to 4, or re-

sponse may be expressed by a continuous variable.13 Inmost systems, a pCR requires the absence of invasive car-cinoma in the breast. Residual ductal carcinoma in situmay be present, as this finding should not alter survival.31

In systems that include lymph nodes, the nodes must also be free of carcinoma for a pCR. The issue of appropriateclassification of ‘‘isolated tumor cells’’ as ‘‘node negative’’or ‘‘node positive’’ after treatment has not been specifi-cally addressed by any study.

Below are listed some of the systems with a brief de-scription on the classification and categories of responses.

AJCC System (Sixth Edition)

Carcinomas are assigned a posttreatment T and N cat-

egory indicated by the prefix ‘‘y.’’ The posttreatmentAJCC (American Joint Committee on Cancer) stage retainsprognostic information but relies predominantly on infor-mation about tumor size and lymph node status, whichcan be difficult to evaluate after NAT.12 Carey et al12 de-fined the T category for AJCC staging system as follows:‘‘When tumor shrinkage from chemotherapy resulted innests of residual tumors, these tumors were categorized by the distance over which the nests extended unless therewere clearly defined multicentric tumor foci that could bedistinguished pathologically.’’ Therefore, this systemequates large contiguous carcinomas with microscopic foci

scattered in a tumor bed. This system does not includechanges in cellularity or lymphovascular invasion (LVI).The posttreatment ‘‘y’’ AJCC stage, by itself, does not givean indication of the response to treatment.

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Table 2. Comparison of Classification Systems for Breast Cancer Response to Therapy and Outcome*

Source, y Name of SystemNo. of

Patients Type of Treatment pCR, %Validated as Prognostic

for Survival

Fisher et al,2 2002 NSABP B-18 1234 AC 20 OS, DFSOgston et al,10 2003 M-P grade 176 CA, vincristine 14 OS, DFSChevallier et al,7 1993 Chevallier 45 FEC-HD (inflammatory cancer) 26 OS, DFS (class 1 and 2 vs 3 and 4)Sataloff et al,8 1995 Sataloff 36 CAF 28 DRFS for T-A vs other categoriesSymmans et al,15 2007 RCB 432 FAC† 16 DRFS

T/FAC‡ 24Carey et al,12 2005 AJCC 132 2 trials:

A → CMFAC → T trastuzumab

17 OS, DFS

* pCR indicates pathologic complete response; NSABP, National Surgical Adjuvant Breast and Bowel Project; AC, doxorubicin and cyclophos-phamide; OS, overall survival; DFS, disease-free survival; M-P, Miller-Payne; CA, cyclophosphamide and doxorubicin; FEC-HD, fluorouracil, epi-rubicin, and cyclophosphamide (high dose); CAF, cyclophosphamide, doxorubicin, and fluorouracil; DRFS, distant relapse–free survival; T-A,Sataloff classification of tumor; RCB, residual cancer burden; FAC, fluorouracil, doxorubicin, and cyclophosphamide;T, paclitaxel; AJCC, American Joint Committee on Cancer; A, doxorubicin; CMF, cyclophosphamide, methotrexate, and fluorouracil.

† 189 patients received FAC treatment.‡ 243 patients received T/FAC treatment.

→

Figure 1. After neoadjuvant chemotherapy, a mastectomy specimen demonstrating the grossly visible fibrotic tumor bed without grossly identifiable residual carcinoma.

Figure 2. After neoadjuvant chemotherapy, a partial mastectomy specimen demonstrating the grossly visible fibrotic tumor bed (2.0 cm) withsmall tan-pink residual tumor nodules. Note that the original pretreatment tumor was 3.0 cm.

Figure 3. Microscopic appearance of the tumor bed after neoadjuvant chemotherapy. A, Stromal fibroelastosis and chronic inflammatory cell infiltrate characterize the tumor bed (hematoxylin-eosin, original magnification 10). B, Tumor bed with scattered hemosiderin-laden macrophages and lymphocytes (hematoxylin-eosin, original magnification 10). C, Tumor bed with large sheets of foamy histiocytes forming nodular aggregates (inset) (hematoxylin-eosin, original magnification 40) and elastosis (hematoxylin-eosin, original magnification 10). D, Chronic inflammatory cells and giant cell reaction to cholesterol clefts within the tumor bed from a case with complete pathologic response (hematoxylin-eosin, original magnification 10).

NSABP B-18 System

This was a system used for one of the largest studiescomparing neoadjuvant to adjuvant therapy.2 Three cate-gories of responses were recognized with only 1 categoryof partial response. Partial response was defined as pres-ence of sparse invasive tumor. Metastases to lymph nodeswere analyzed separately. The study was correlated withoverall survival and disease-free survival.

The Miller-Payne System

Response was divided into 5 grades based on a com-parison of tumor cellularity before and after treatment andwas correlated with disease-free and overall survival.10

This study showed that a grade-4 response (almost a pCR)had a worse prognosis than a pCR (grade 5), providingevidence that this type of response should be kept as aseparate group. However, this system does not include theresponse in lymph nodes or the presence of LVI. Thus, it

is possible that the patients with a grade 4 response whodid poorly either had lymph node metastases or had LVI.

Chevallier System

This system classified the treatment response of 45women with inflammatory carcinoma into 4 groups with1 category of partial response.7 The authors separated cas-es with residual ductal carcinoma in situ from cases withno residual carcinoma. In most systems, these are bothgrouped together as pCR. In this system, partial responsecategory was combined with the no response category foroutcome analysis.

Sataloff System

This system classified treatment response in 36 patientsfor both the primary carcinoma and the lymph nodes.8

The category of pCR (called T-A) includes a ‘‘near totaltherapeutic effect,’’ implying that small foci of invasivecarcinoma may be present. There are 2 categories of par-tial response. This system does not include LVI. The studyshowed that patients with T-A had a better survival at 5years than the other 3 categories of patients, but did notshow differences in the categories of partial response.

RCB System

This system was developed to calculate residual cancer burden (RCB) in 382 patients in 2 different treatment co-horts for prediction of distant relapse–free survival.15 Thissystem uses residual invasive carcinoma cellularity dis-tributed over the tumor bed, the number of lymph nodeswith metastases, and the size of the largest metastasis

combined mathematically into a continuous index to de-fine 4 categories of RCB (RCB-0 through RCB-III). Al-though the system requires the use of a formula, a Web- based calculation script is freely available to calculate thescores (http://www.mdanderson.org/breastcancer RCB).

In addition to typical neoadjuvant therapy (NAT) inwhich chemotherapy is completed before surgery, someprotocols may involve short-term therapy (eg, weeks in-stead of months) between diagnosis and definitive sur-gery. Alternatively, therapies based on NAT protocols us-ing only hormonal agents may need to be of longer du-ration to develop a maximal response. Different classifi-

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cation schemes for tumor response will be developed astreatments are used in different ways.

PATHOLOGIC EXAMINATION AFTERNEOADJUVANT THERAPY

Tumor response can be evaluated clinically by palpationor by breast imaging, and these are useful techniques tomonitor response during therapy. However, clinical andradiologic assessments of response often underestimate oroverestimate the amount of residual carcinoma present.

Thus, pathologic examination of the excised tumor bed isthe gold standard and is essential for identifying thegroup of patients (typically 15% to 28%) with a pathologiccomplete response to treatment, as well as the other 60%to 70% of patients who have a partial response to treat-ment.2,7–14

The Pretreatment Specimen

Typically, the pretreatment specimen will be a core nee-dle biopsy. Because 15% to 28% of patients will have noresidual tumor after NAT, it is important to have an ade-quate pretreatment sample in which an unequivocal di-agnosis of invasive carcinoma is established and evalua-tion of hormone receptors and HER2/neu status is com-

pleted before treatment. Predictors of a pCR include highhistologic grade, nonlobular histologic type, estrogen re-ceptor negativity, extensive tumor necrosis, and HER2/neu overexpression (for patients who would receive treat-ment targeting this antigen).32 It is therefore helpful to in-clude histologic type, grade, minimal size, presence of tu-mor necrosis, and LVI to enable comparison of thesefeatures to the posttreatment carcinoma.

The Posttreatment Specimen

It is important for the pathologist to be aware of the useof neoadjuvant therapy before examining a pathologicspecimen. Unfortunately, this history is not always provid-ed. Important clues are a long interval between a priorcore needle biopsy and a subsequent excision (typically

months) and/or a specimen without an obvious biopsycavity or a clearly palpable carcinoma. The following clin-ical information should be provided to the pathologist.

1. Presentation of the lesion before treatment (palpablemass or radiologic lesion, skin changes such as edema,erythema, fixation to chest wall)

2. Size of the invasive carcinoma before treatment3. Prior diagnostic procedure: core needle biopsy or in-

cisional biopsy—ideally, this specimen should be availablefor comparison with the posttreatment carcinoma

4. Presence of a clip and/or calcifications in the tumor5. Prior evaluation of lymph nodes (fine-needle aspira-

tion, core biopsy, or sentinel lymph node biopsy) and theresults

6. Type of neoadjuvant therapy7. Clinical/radiologic response of the carcinoma to the

treatment (complete, partial, minimal)

Primary Tumor Specimen. Identification of the TumorBed. It is imperative to identify the original tumor (tu-mor bed) to determine whether or not the patient has hada pCR and to allow localization for breast preservation. If the patient had a complete response clinically and radio-logically, it is unlikely a gross lesion will be present. How-ever, microscopic examination in such cases may revealsubstantial residual tumor. Therefore, for patients who re-

ceive NAT, a clip should be placed during a diagnosticcore needle biopsy or at the first few cycles of therapy,ideally before the tumor is too soft or unrecognizable onimaging studies. If a clip is not placed, it may be impos-sible to reliably identify the tumor bed. When calcifica-tions are associated with the carcinoma, they usually re-main after treatment and can be detected on a specimenradiograph. Ideally, candidates who qualify for breast-con-serving surgery after NAT should undergo wire/needlelocalized excisional biopsy. It is prudent to obtain a spec-

imen radiograph demonstrating a clip or calcifications todocument that the tumor bed was excised. Lacking these,one must rely on the surgeon and/or microscopic detec-tion of the tumor bed to ensure that the prior tumor sitewas removed, especially when no gross lesion is visible.For mastectomy specimens without a radiologically iden-tifiable lesion, a detailed description of the pretreatmenttumor location (eg, quadrant, distance from nipple) andsutures placed by the surgeon are extremely helpful infinding the tumor bed.

Grossly, the tumor bed may have the appearance of asubtle irregular area of rubbery fibrous tissue (Figure 1).Residual tumor may be recognized as fleshy noduleswithin the tumor bed (Figure 2). It is important to docu-

ment the size of the grossly visible tumor bed and anyrecognizable residual tumor. In some cases, the tumor bedmay not be recognizable grossly. For patients who under-go breast-conserving surgery, the specimens should bedifferentially inked to assess margins in the event residualtumor is found on microscopic examination.

Sampling of Tumor Bed. The degree to which a residualtumor bed should be sampled has not been established.For initial sampling, 1 block per centimeter of pretreat-ment tumor size is reasonable. If residual tumor is found,additional sampling is not necessary. If no tumor is found,it is not yet known how much additional sampling isneeded. However, if the residual tumor bed is small, theentire area should be submitted for pathologic analysis. If the residual tumor is still large (5 cm), then one should

consider examining at least 5 representative sections fromthe largest cross-sectional area. The Web site that offersthe residual cancer burden calculator (http://www.mdanderson.org/breastcancer RCB) has some recommen-dations on tumor sampling.

Microscopic Appearance of Tumor Bed. The presence of atumor bed must be confirmed microscopically when re-sidual carcinoma is not present. Microscopically, the tu-mor bed is characterized by an area of hyalinized vascularstroma with stromal edema and fibroelastosis, often with-out the presence of normal glandular breast ducts and lob-ules (Figure 3, A). The stroma is often infiltrated by foamyhistiocytes, sometimes forming large sheets, aggregates of lymphocytes, and hemosiderin pigment (Figure 3, B and

C). Areas of tumor necrosis may leave nodules of histio-cytes and cholesterol clefts (Figure 3, D).The cytologic features of most carcinomas do not

change after treatment except for the decrease in cellular-ity. However, some carcinomas (Figure 4, A) show changesindicative of treatment effect (Figure 4, B). These changesinclude distortion of glandular architecture, enlarged tu-mor cells due to increased cytoplasm, cytoplasmic vacu-olization and eosinophilic change, pleomorphic and bi-zarre nuclei, and decreased mitotic activity (Figure 4, B,inset). Residual tumor cells are often distributed eithersingly or in clusters; in the latter case, the borders are

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Figure 4. An infiltrating ductal carcinoma with partial response and cytologic changes due to treatment effect of chemotherapy. A, Pretreatment core biopsy of a poorly differentiated infiltrating carcinoma with frequent mitoses (inset) (hematoxylin-eosin, original magnification 60) and tumor necrosis (hematoxylin-eosin, original magnification 10). B, Same tumor after treatment exhibiting marked epithelial atypia, tumor giant cell formation, cytoplasmic eosinophilia, and pleomorphic nuclei. The tumor cell groups show stromal retraction artifact (hematoxylin-eosin,original magnification 10). Note that no mitotic figures are identified after treatment (inset) (hematoxylin-eosin, original magnification 60).

Figure 5. An infiltrating lobular carcinoma with partial pathologic response to neoadjuvant chemotherapy. A, An infiltrating lobular carcinomademonstrating marked cellularity on a core needle biopsy before treatment (hematoxylin-eosin, original magnification 10). B, An almost complete pathologic response after treatment of the tumor in (A). The residual tumor cells are sparse and scattered as single cells with hyperchromatic nuclei in a fibroelastotic stroma (hematoxylin-eosin, original magnification 10). The multinucleated tumor cells show intracytoplasmic mucin, cytoplasmic vacuolization, and nuclear smudging (inset) (hematoxylin-eosin, original magnification 60).

typically well defined and the cells tend to shrink awayfrom the stroma (Figure 4, B). This feature should not bemisinterpreted as lymphovascular invasion. In cases of near complete pathologic response, scattered single de-generated tumor cells may show multinucleation, hyper-chromasia, and nuclear smudging, making them difficultto detect on routine hematoxylin-eosin stain (Figure 5, Aand B). In difficult cases, immunohistochemical stains todistinguish between epithelial cells (cytokeratins AE1/AE3 or cytokeratin 7) and histiocytes (CD68 or CD163)are helpful in identifying the residual tumor cells, as wellas in the evaluation of surgical margins. Residual ductal

carcinoma in situ usually does not show morphologic al-teration after treatment.Changes in Normal Breast. Cytotoxic treatment effect

also occurs in the nontumor–bearing breast parenchymain the form of moderate to marked sclerosis of basementmembranes of the ductal and acinar components of theterminal duct–lobular unit. Scattered epithelial cells in thisunit may show cytologic and nuclear enlargement (Figure6), which should not be confused with residual in situcarcinoma.

Evaluation of Margins. Margins can be more difficult toevaluate after neoadjuvant therapy (NAT). It is preferable

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Figure 6. Terminal duct–lobular unit in the nonneoplastic breast after neoadjuvant chemotherapy with therapy effect in the form of atrophy,hyalinized basement membrane, and scattered, cytologically atypical epithelial cells (hematoxylin-eosin, original magnification 10).

to excise the entire tumor bed with a rim of normal tissueto ensure there is no residual carcinoma. However, theextent of the tumor bed can be difficult to determine onimaging by the radiologist, grossly by the surgeon, andmacroscopically by the pathologist when there has been apronounced response to treatment. The significance of tu-mor bed at the margin is unclear in patients with a pCR.In cases with scattered residual foci of invasive carcinomaor ductal carcinoma in situ throughout a tumor bed, tu-mor bed changes at the margin may be predictive of thepossibility of residual carcinoma in the breast.

Posttreatment Lymph Node Evaluation. The axillarytail should be carefully searched for lymph nodes and all

nodes thinly sectioned and completely submitted. In gen-eral, after NAT, lymph nodes are difficult to recognize be-cause of atrophy and fibrosis. When it is difficult to locatelymph nodes, fibrotic areas in the axillary fat and tissuearound the vessels should be submitted, which may revealsmall atrophic nodes on microscopic examination.

Patients who have a positive lymph node by fine-needleaspiration (or core needle biopsy) before neoadjuvant ther-apy usually undergo completion axillary dissection at thetime of primary tumor resection. This approach helps tostratify patients into 3 groups, namely those with (1) pos-itive nodes, with or without evidence of disease regression(partial or no response); (2) negative nodes, with evidenceof treatment-induced change but no viable tumor (com-

plete response); and (3) negative nodes without treatmenteffect (complete response). If a positive lymph node wasremoved before therapy, and the lymph nodes after ther-apy are not involved by metastases, response to therapycannot be evaluated with certainty.

Lymph node metastases that show complete responseto treatment are often replaced by hyaline stromal scars,mucin pools, or aggregates of histiocytes without any vi-able tumor cells (Figure 7, A and B). Complete pathologicresponse to prior metastatic involvement in some casescannot be determined with certainty because metastasiscan resolve without a scar or may leave small fibrous

scars. However, it is unusual to see large fibrous scars inlymph nodes of patients who undergo surgery first.33

Therefore, the presence of a large scar in a lymph nodewithout tumor cells most likely is indicative of a completeresponse to therapy.

Partial response in lymph nodes is characterized by iso-lated or clusters of tumor cells surrounded by thin or thickhyaline stromal fibrosis (Figure 8, A and B). Immunohis-tochemical stains (cytokeratins) can be used to identifytumor cells that are difficult to characterize on routine he-

matoxylin-eosin stain. Patients who have residual meta-static tumor with evidence of treatment effect have betterdisease-free survival and lower relapse rates than patientswho have positive nodes without evidence of such chang-es.34 Therefore, it is important to make notation of treat-ment effect in lymph nodes.

REPORTING OF BREAST CARCINOMAS AFTERNEOADJUVANT THERAPY

Pathology reports on treated tumors should include thefollowing information.

Breast Specimen

1. Presence and size of tumor bed: important for doc-

umentation, especially in cases with pathologic completeresponse

2. Size and extent of residual tumorTwo-dimensional measurements of the largest area

of invasive cancerNumber of foci or number of blocks with foci of in-

vasion3. Average cancer cellularity of the residual tumor bed

(see Table 1 for Miller-Payne grading system, which re-quires evaluation of the change in cellularity and for RCBsystem, which has examples and guidelines to assess re-sidual cellularity)

4. Appearance of the residual tumor and grade, if ap-plicable: compare to pretreatment carcinoma, if possible

5. Viability (necrosis, mitotic figures); proliferation in-dex by MIB-1 (Ki-67) may be requested for some protocols6. Lymphovascular invasion7. Presence and extent of ductal carcinoma in situ (per-

centage of in situ component when using the RCB system)8. Margins with respect to tumor bed, invasive, and in

situ carcinoma9. A comment on the overall response to treatment

Lymph Nodes

1. Number of lymph nodes2. Number of lymph nodes with metastases3. Size of the largest metastasis4. Presence of extranodal extension (measurement of

largest extent of extranodal extension may be requested by some radiation oncologists)

5. Number of metastases with evidence of treatment re-sponse

6. Number of lymph nodes with evidence of treatmentresponse but without tumor cells (ie, fibrosis, necrosis, ag-gregates of histiocytes)

Classification of Response

1. By AJCC staging, pT category and pN category as-signed a prefix ‘‘y’’ (‘‘p’’ refers to pathologic classification)

2. Response category according to 1 of the classification

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Figure 7. Axillary lymph node with complete pathologic response after neoadjuvant chemotherapy. A, The node is replaced by large sheets of foamy histiocytes and giant cells with no residual tumor (hematoxylin-eosin, original magnification 10). B, Axillary lymph node from a different case with pathologic complete response. The metastatic tumor is completely replaced by fibro-collagenized stroma (hematoxylin-eosin, original magnification 10). A fine-needle aspiration biopsy of these node before treatment contained metastatic carcinoma.

Figure 8. Residual tumor (partial response) in axillary lymph node after neoadjuvant chemotherapy with treatment effects. A, A sentinel lymphnode biopsy specimen demonstrating metastatic lobular carcinoma before chemotherapy (hematoxylin-eosin, original magnification 10). B, Apositive axillary node from the same patient showing residual hyperchromatic tumor cells embedded in a fibrous stroma after treatment (hematoxylin-eosin, original magnification 10).

systems as used by specific institutions or for clinical pro-tocols

CONCLUSIONSNeoadjuvant therapy is being offered more commonly

to patients with earlier-stage breast cancer and is likely to become the standard of care for patients receiving system-ic therapy. Because clinical and radiologic responses donot correlate well with residual tumor after treatment,pathologic evaluation of tumor response is the gold stan-dard. Pathologists have played, and will continue to play,an important role in providing this information to opti-mize the knowledge gained by this approach to breastcancer therapy. The role of pathologists is vital in stan-

dardizing the existing classification schemes and in de-veloping new schemes for ongoing trials.

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